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PubMed1995

Hyperpigmentation and melanocytic hyperplasia in transgenic mice expressing the human T24 Ha-ras gene regulated by a mouse tyrosinase promoter.

The tyrosinase promoter has been used to target expression of the mutated human T24 Ha-ras oncogene in pigment-producing cells of transgenic mice. Two independent founder mice carrying the transgene survived and showed the same distinct phenotype of mutated coat color, deeply pigmented skin with multiple nevi, and twirling behavior. The offspring of one of these founders were developed into a line that stably expressed the same phenotype. Histopathological analysis of the tissues revealed hyperpigmentation and/or melanocytic hyperplasia in the skin, eyes, inner ear, and meningeal membranes in the brain. Reverse transcriptase-polymerase chain reaction analysis revealed expression of the transgene in skin, brain, and spleen. We propose that these transgenic mice will be a model for studying the process of multistage melanoma carcinogenesis and a system for evaluating potential chemopreventive agents.

Animals↗
PubMed2007

A promoter element with enhancer properties, and the orphan nuclear receptor RORalpha, are required for Purkinje cell-specific expression of a Gi/o modulator.

The promoter and structural portion of the gene, Pcp-2(L7), has frequently been used to target expression of proteins to cerebellar Purkinje cells. In our continuing analysis of the transcription of this gene and how it relates to the G-protein and Ca2+ channel modulatory functions of the encoded protein, we have dissociated the promoter and structural gene and identified cooperative functions. A 0.9 kb fragment of the proximal promoter has positional properties of a classical enhancer, yet its function requires the presence of the structural gene. We demonstrate that RORalpha, the gene product of the mutant mouse locus called staggerer (Rora(sg)), binds to and activates expression through this promoter element using functional assays in vitro and in vivo. The structural gene has a repressive effect on gene expression outside Purkinje cells, and likely participates in the suppression of Pcp-2(L7) gene expression in the many other brain and non-neuronal cell types, besides Purkinje cells, known to express RORalpha. Additional studies in vivo show that while Pcp-2(L7) expression is dependent on RORalpha throughout the cerebellum, this dependence is greatest in the intermediate region between the vermis and far lateral hemispheres. Thus, in addition to its recently indicated role in Ca2+-mediated reciprocal cell-cell signaling in Purkinje cells, RORalpha may also contribute to functional differences in cerebellar subregions.

Animals↗
PubMed2003

Spatiotemporal rescue of memory dysfunction in Drosophila.

We have developed a method for temporal and regional gene expression targeting (TARGET) in Drosophila and show the simultaneous spatial and temporal rescue of a memory defect. The transient expression of the rutabaga-encoded adenylyl cyclase in the mushroom bodies of the adult brain was necessary and sufficient to rescue the rutabaga memory deficit, which rules out a developmental brain defect in the etiology of this deficit and demonstrates an acute role for rutabaga in memory formation in these neurons. The TARGET system offers general utility in simultaneously addressing issues of when and where gene products are required.

Adenylyl Cyclases↗
PubMed1995

Restricted tissue-specific but correct developmental expression mediated by a short human alpha 1AT promoter fragment in transgenic mice.

The tissue-specific and developmental pattern of expression controlled by the proximal promoter (position-348 to +15) derived from the human alpha-1-antitrypsin (h alpha 1AT) gene was studied in transgenic mice. The short promoter segment was linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The transgene showed highly specific expression in the liver and the correct developmental pattern of regulation. Interestingly, this short promoter targets expression to the liver with a greater specificity than that reported for larger alpha 1AT promoter fragments.

Animals↗
PubMed2003

In vivo gene transfer studies on the regulation and function of the vasopressin and oxytocin genes.

Novel genes can be introduced into the germline of rats and mice by microinjecting fertilized one-cell eggs with fragments of cloned DNA. A gene sequence can thus be studied within the physiological integrity of the resulting transgenic animals, without any prior knowledge of its regulation and function. These technologies have been used to elucidate the mechanisms by which the expression of the two genes in the locus that codes for the neuropeptides vasopressin and oxytocin is confined to, and regulated physiologically within, specific groups of neurones in the hypothalamus. A number of groups have described transgenes, derived from racine, murine and bovine sources, in both rat and mouse hosts, that mimic the appropriate expression of the endogenous vasopressin and genes in magnocellular neurones (MCNs) of the supraoptic and paraventricular nuclei. However, despite considerable effort, a full description of the cis-acting sequences mediating the regulation of the vasopressin-oxytocin locus remains elusive. Two general conclusions have nonetheless been reached. First, that the proximal promoters of both genes are unable to confer any cell-specific regulatory controls. Second, that sequences downstream of the promoter, within the structural gene and/or the intergenic region that separates the two genes, are crucial for appropriate expression. Despite these limitations, sufficient knowledge has been garnered to specifically direct the expression of reporter genes to vasopressin and oxytocin MCNs. Further, it has been shown that reporter proteins can be directed to the regulated secretory pathway, from where they are subject to appropriate physiological release. The use of MCN expression vectors will thus enable the study of the physiology of these neurones through the targeted expression of biologically active molecules. However, the germline transgenic approach has a number of limitations involving the interpretation of phenotypes, as well as the large cost, labour and time demands. High-throughput somatic gene transfer techniques, principally involving the stereotaxic injection of hypothalamic neuronal groups with replication-deficient adenoviral vectors, are now being developed that obviate these difficulties, and which enable the robust, long-lasting expression of biologically active proteins in vasopressin and oxytocin MCNs.

Animals↗
PubMed2002

Calmodulin regulates the transcriptional activity of estrogen receptors. Selective inhibition of calmodulin function in subcellular compartments.

The steroid hormone estrogen elicits biological effects in cells by binding to and activating the estrogen receptor (ER). Estrogen binding induces a conformational change in the receptor, inducing nuclear translocation and transcriptional activation of ER. The ubiquitous Ca(2+)-binding protein calmodulin has been shown to interact directly with ER and enhance its stability. To further elucidate the functional sequelae of the association between calmodulin and ER, we examined the effect on ER transcriptional activation of specifically inhibiting calmodulin. The cell-permeable calmodulin antagonist CGS9343B prevented estrogen-induced transcriptional activation by ER, without altering basal transcription. The inhibition was dose-dependent and independent of the time of estrogen stimulation. To validate these findings, calmodulin function was also neutralized by targeted expression of a specific inhibitor peptide. By inserting localization signals, the inhibitor peptide was selectively targeted to different subcellular domains. Inactivation of calmodulin function in the nucleus virtually eliminated estrogen-stimulated ER transcriptional activation. By contrast, when membrane calmodulin was specifically neutralized, estrogen-stimulated transcriptional activation by ER was only slightly attenuated. Importantly, the inhibitor peptides did not significantly reduce the amount of ER in the cells. Together, these data demonstrate that calmodulin is a fundamental component of ER transcriptional activation.

Animals↗
PubMed2005

Normal lung development in RAIG1-deficient mice despite unique lung epithelium-specific expression.

RAIG1, 2, and 3 and GPCR5d represent a new subfamily of orphan G protein-coupled receptors. RAIG1 is expressed abundantly and specifically in the lung during development and in adult mice. During lung development, RAIG1 expression is initiated at E14.5 and gradually increases, reaching its highest levels at E18. High levels of expression are maintained in adult lungs. Given its abundant lung-specific expression and role in retinoic acid signaling, we hypothesized that RAIG1 plays a role in epithelial cell differentiation during lung development. To determine RAIG1 function and track endogenous RAIG1 spatial expression, a null allele of Raig1 was generated and the lacZ gene was "knocked-in." Although expression was detected in both proximal and distal epithelium during embryogenesis, it became restricted to type I and type II pneumocytes and the most distal bronchiolar cells in postnatal lungs. This is the first gene known to have this unique epithelial cell expression pattern. Despite this high level of expression, targeted inactivation of Raig1 did not cause significant developmental defects. Epithelial cell differentiation was normal and lung structure was intact. Analysis of other family members demonstrated some overlapping embryonic expression of RAIG3 mRNA that could have led to functional redundancy in the single RAIG1 null mutant mouse.

Animals↗
PubMed2002

Antigen presenting cells transfected with LMP2a RNA induce CD4+ LMP2a-specific cytotoxic T lymphocytes which kill via a Fas-independent mechanism.

Recent reports have demonstrated that EBV can be used as a target of specific CTL-based treatments in severe chronic EBV, immunoblastic B cell lymphoma and Hodgkin's disease (HD). Based upon the promising results form these in vivo studies, it has been suggested that an antigen-specific CTL-based immunotherapy may be of benefit in treating EBV-associated tumors such as HD and nasopharyngeal carcinoma (NPC) which express the potentially immunogenic antigens, LMP1 and LMP2a. Recent work form our group has demonstrated that LMP2a-specific CTLs may be generated in vitro using autologous antigen presenting cells which have been transfected with polyadenylated LMP2a RNA in the presence of a cationic lipid. In this study, we demonstrate that the presence of the lipid enhances dendritic cell (DC) transfection efficiency and appears to protect the intracellular LMP2a RNA from degradation by cellular RNAses. Significantly, these improvements resulted in the transfected DCs having a superior ability to stimulate autologous T cell proliferation. These LMP2a + DCs were used to stimulate LMP2a-specific effector cells which were predominantly a mixture of cytotoxic and helper CD4+ T cells. The molecular mechanisms whereby these CD4+ T cells lyzed their LMP2a-expressing targets was investigated and we show that, although expressing Fas ligand on their surface, LMP2a-specific CD4+ effector cells kill their targets using the Ca2+-dependent perforin/granzyme pathway which is the same mechanism used by CD8+ CTLs.

Anti-Bacterial Agents↗
PubMed2003

Application of transgenesis in livestock for agriculture and biomedicine.

Microinjection of foreign DNA into pronuclei of a fertilized oocyte has predominantly been used for the generation of transgenic livestock. This technology works reliably, but is inefficient and results in random integration and variable expression patterns in the transgenic offspring. Nevertheless, remarkable achievements have been made with this technology. By targeting expression to the mammary gland, numerous heterologous recombinant human proteins have been produced in large amounts which could be purified from milk of transgenic goats, sheep, cattle and rabbit. Products such as human anti-thrombin III, alpha-anti-trypsin and tissue plasminogen activator are currently in advanced clinical trials and are expected to be on the market within the next few years. Transgenic pigs that express human complement regulating proteins have been tested in their ability to serve as donors in human organ transplantation (i.e. xenotransplantation). In vitro and in vivo data convincingly show that the hyperacute rejection response can be overcome in a clinically acceptable manner by successful employing this strategy. It is anticipated that transgenic pigs will be available as donors for functional xenografts within a few years. Similarly, pigs may serve as donors for a variety of xenogenic cells and tissues. The recent developments in nuclear transfer and its merger with the growing genomic data allow a targeted and regulatable transgenic production. Systems for efficient homologous recombination in somatic cells are being developed and the adaptation of sophisticated molecular tools, already explored in mice, for transgenic livestock production is underway. The availability of these technologies are essential to maintain "genetic security" and to ensure absence of unwanted side effects.

Agriculture↗
PubMed2001

Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype.

Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex in Hyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalized Phex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phex transgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phex function in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect the Phex substrate FGF-23 in osteoblasts. Taken together, these in vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites of Phex expression and/or additional factors are likely to be important in the pathogenesis of XLH.

Alkaline Phosphatase↗
PubMed2000

A non-cleavable mutant of Fas ligand does not prevent neutrophilic destruction of islet transplants.

BACKGROUND: Fas ligand (FasL) mediates apoptosis of susceptible Fas-expressing lymphocytes, and may contribute to the maintenance of peripheral tolerance. In transplantation models, however, artificial expression of FasL on cellular as well as islet transplants results in accelerated rejection by neutrophils. The mechanism of the neutrophilic response to FasL expression is unknown. FasL, like other members of the tumor necrosis factor family, is cleaved to a soluble form by metalloproteases. We tested the hypothesis that soluble FasL (sFasL) was responsible for neutrophil migration by creating a non-cleavable mutant of FasL. METHODS: Three mutants of FasL with serial deletions in the putative proteolytic cleavage site of human FasL were made using inverse polymerase chain reaction. The relative fractions of sFasL and membrane-bound FasL were assessed by Western blot and immunoprecipitation, as well as by cytotoxicity assay using Fas-expressing target cells. The fully non-cleavable mutant was transduced into murine islets as well as myoblasts and tumor cell lines, and tested in a murine transplantation model. RESULTS: Serial deletions in the putative metalloprotease site of FasL resulted in a fully non-cleavable mutant of FasL (ncFasL). Expression of ncFasL in tumor lines induced higher levels of apoptosis in Fas bearing targets than wild-type FasL. Transplantation of ncFasL-expressing islets under the kidney capsule of allogenic mice resulted in accelerated rejection identical to that seen with wild-type Fas ligand-expressing islets. Myoblasts and tumor cell lines expressing ncFasL also induced neutrophil infiltration. CONCLUSIONS: Membrane-bound Fas ligand is fully capable of inducing a neutrophilic response to transplants, suggesting an activation by Fas ligand of neutrophil chemotactic factors.

Animals↗
PubMed1991

Induction of HIV-specific CTL and antibody responses in mice using retroviral vector-transduced cells.

Recombinant retroviral vectors can efficiently transduce and express foreign genes in mammalian cells. We have examined the utility of retroviral vector-mediated gene transfer to deliver genes which encode human immunodeficiency virus type I (HIV) antigens capable of stimulating specific immune responses. Murine fibroblast cell lines were transduced with a nonreplicating murine retroviral vector carrying the gene encoding the HIV-IIIB envelope protein and were shown to express the gp160/120 protein. Mice immunized with syngeneic vector-transduced cells developed CD8+, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) specific for targets expressing the HIV envelope protein. The CTL also exhibited lytic activity on target cells coated with synthetic peptides derived from the gp120 V3 hypervariable region of both the HIV-IIIB and HIV(MN) isolates. Following adoptive transfer in a murine tumor model, these CTL were shown to be effective in vivo by their ability to eliminate established tumor cells expressing the HIV protein. Vector-transduced syngeneic cells were also capable of eliciting HIV envelope-specific antibody responses in immunized mice. Sera obtained from these mice were found to bind to the HIV-IIIB gp160 protein as well as a peptide-defined neutralizing antibody epitope contained within the V3 domain of gp120. These sera exhibited virus-neutralizing activity in that they markedly reduced the ability of HIV to infect and form syncytia of a human T-cell line. This is the first demonstration that cells transduced with a retroviral vector encoding the HIV-IIIB envelope protein are capable of inducing effective HIV-specific cellular and humoral immune responses in mice.

Amino Acid Sequence↗
PubMed2002

Overexpression of a constitutively active form of c-src in skin epidermis increases sensitivity to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

Transgenic mice were developed to study the role of c-src in epithelial tumorigenesis through targeted expression of a constitutively active form of murine c-src (src(529)). Src(529) was targeted to the interfollicular epidermis with the human keratin 1 (HK1) promoter. The skin phenotype of these mice was characterized by exaggerated epidermal hyperplasia and hyperkeratosis within the first week after birth. The severity of this phenotype correlated with overall src kinase activity, both of which subsided with age. Treatment of adult HK1.src(529) transgenic mice with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate resulted in an increase in epidermal hyperplasia and labeling index significantly greater than that seen in nontransgenic littermates. In addition, HK1.src(529) transgenic mice developed papillomas earlier and in significantly greater numbers compared with nontransgenic littermates in a standard initiation-promotion experiment. The data support the hypothesis that activation of c-src kinase plays a role in skin tumor promotion.

Animals↗
PubMed2004

Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes.

We report the differentiation of human adipose tissue stem cells (ATSCs) to take on cardiomyocyte properties following transient exposure to a rat cardiomyocyte extract. Reversibly permeabilized ATSCs were incubated for 1h in a nuclear and cytoplasmic extract of rat cardiomyocytes, resealed with CaCl(2), and cultured. Three weeks after exposure to extract, ATSCs expressed several cardiomyocyte markers including sarcomeric alpha-actinin, desmin, and cardiac troponin I, and displayed targeted expression of the gap junction protein connexin 43. Formation of binucleated and striated cells, and spontaneous beating in culture were also observed. A low proportion of intact ATSCs exposed to the extract also showed signs of alpha-actinin and connexin 43 expression. Additional evidence of differentiation was provided by induction of expression of nuclear lamin A/C, a marker of terminally differentiated cells, and a remarkable increase in cell cycle length. Together with our previous data, this study suggests that alteration of cell fate using cellular extracts may be applied to multiple cell types. Cell extracts may also prove useful for investigating the molecular mechanisms of stem cell differentiation.

Actinin↗
PubMed2006

MEKK1 controls matrix degradation and tumor cell dissemination during metastasis of polyoma middle-T driven mammary cancer.

Mammary tumor cells are required to degrade the surrounding matrix and disseminate in order to metastasize, and both of these processes are controlled by a tumor cell-signaling network that remains poorly defined. MEKK1 is a MAPKKK that regulates both the extracellular signal regulated kinase (ERK1/2) and the c-Jun amino terminal kinase (JNK) signaling pathways. MEKK1 signaling regulates migration through control of cell adhesion and is required for inducible expression of urokinase-type plasminogen activator (uPA). MEKK1-deficient mice with mammary gland-targeted expression of the polyoma middle T antigen (PyMT) transgene develop primary mammary tumors at a rate and frequency similar to wild-type littermates, indicating that MEKK1 deficiency does not affect PyMT-mediated transformation. However, MEKK1-/- mice display significantly delayed tumor cell dissemination and lung metastasis. Delayed MEKK1-dependent tumor dissemination is associated with markedly reduced tumor uPA expression, gelatinase activity, and prolonged tumor basement membrane integrity. siRNA-mediated MEKK1 knockdown inhibits uPA activity, cell migration and invasion in MDA-MB-231 human breast cancer cells. Thus MEKK1 controls tumor progression by regulating both the migration and proteolysis aspects of tumor cell invasiveness. To our knowledge, this is the first example of a MAPKKK that regulates metastasis through control of tumor invasiveness.

Animals↗
PubMed2000

Enhanced cardiomyocyte DNA synthesis during myocardial hypertrophy in mice expressing a modified TSC2 transgene.

Tuberous sclerosis complex (TSC) is a rare genetic disorder characterized by the appearance of benign tumors in multiple organs, including the heart. Disease progression is accompanied by homozygous mutation at 1 of 2 loci (designated TSC1 or TSC2), leading to the suggestion that these genes function as tumor suppressors. In this study, we generated a series of TSC2 cDNAs in which one or more structural motifs were deleted, with the hope that expression of the modified gene product would override the growth-inhibitory activity of the endogenous TSC2 gene product. Several of the modified cDNAs enhanced growth rate, increased endocytosis, and promoted aberrant protein trafficking when expressed in NIH-3T3 cells, thereby mimicking phenotypes typical of TSC2-deficient cells. Surprisingly, targeted expression of the most potent TSC2 cDNA to the heart did not perturb cardiac development. However, the level of cardiomyocyte DNA synthesis in adult transgenic mice was elevated >35-fold during isoproterenol-induced hypertrophy compared with their nontransgenic siblings. These results suggest that alteration of TSC2 gene activity in combination with beta-adrenergic stimulation can reactivate the cell cycle in a limited number of terminally differentiated adult cardiomyocytes.

3T3 Cells↗
PubMed2003

Transcriptional gene expression profiling of small cell lung cancer cells.

A global gene expression analysis using oligonucleotide microarrays was performed on many human small cell lung cancer (SCLC) cell lines in cell culture and/or as xenografts. The expression was compared with the expression profiles of 18 normal tissues. In a hierarchical cluster analysis the cell lines clustered distinctly from normal tissues and grouped into four clusters. One cluster consisted of two related cell lines and was markedly different from the other SCLC cell lines, whereas the rest of the clusters grouped together. Two subclusters contained the classical SCLC types and one subcluster the variant SCLC type, thus identifying many genes with differential expression between the two variants of SCLC. All of the xenografts clustered closest to the cell lines from which they originated and had the same expression levels as the cells grown in culture for the majority of genes. The analysis confirmed the high expression of many genes identified previously as highly expressed in SCLC cells including neuroendocrine markers, oncogenes, and genes involved in cell proliferation and division. The analysis furthermore identified a number of molecules not identified previously as expressed in SCLC. Several of these are expressed in low or undetectable amounts in the majority of normal tissues and, therefore, are potential targets for new therapeutic approaches. By including the published array profiles of six ressected SCLC tumors from Bhattacharjee et al. (A. Bhattacharjee et al., Proc. Natl. Acad. Sci. USA, 98: 13790-13795, 2001.), the analysis revealed that most of the novel potential targets expressed by SCLC cell lines and xenografts were also expressed in the tumors. This analysis demonstrates the value of using cell lines and xenografts for expression profiling, when a limited quantity of tumor material is available.

Adult↗
PubMedDate not supplied

Interleukin 18 enhances Fas ligand expression and induces apoptosis in Fas-expressing human myelomonocytic KG-1 cells.

Interleukin-18 (IL-18) induces apoptosis in human myelomonocytic KG-1 cells as determined by agarose gel electrophoresis, and flow cytometry after propidium iodide (PI) staining. Apoptosis was detected 20 hours from the start of culture at concentrations of 100 ng/ml of the cytokine. Although IL-18 induces the production of large amounts of interferon gamma (IFN-gamma) by KG-1 cells, conditioned media could not induce apoptosis of fresh cells. The protein expressions of p53 and Fas ligand by KG-1 cells, which constitutively express the Fas antigen (CD95), were found to increase after exposure to IL-18 for 20 hours. Both Fas ligand and its receptor were found to be functional by in vitro assays on Fas-expressing target cells and an agonist anti-Fas antibody, respectively. In conclusion, IL-18 enhances the expression of Fas ligand by Fas-expressing KG-1 cells and induces apoptosis in the cells through a mechanism probably involving the Fas pathway.

Apoptosis↗